The Hippo signaling pathway is a developmentally conserved pathway involved in organ size control, cellular proliferation and differentiation. Multiple lines of evidence suggest that deregulation of Hippo signaling plays a role in pancreatic cancer, include frequent overexpression of hYAP, the nuclear effector of mammalian Hippo signaling, in pancreatic cancer tissues and cell lines, and less commonly, somatic mutations of hFAT1 and hFAT4, human orthologs of a Drosophila cadherin gene involved in Hippo repression and growth suppression. Ectopic hYAP expression in non-tumorigenic human pancreatic epithelial cells transforms these cells, while knockdown of endogenous hYAP by RNA interference in pancreatic cancer lines inhibits in vivo growth in athymic mice. The transcriptional targets of hYAP in human cells include genes associated with invasion, metastases, and tumor angiogenesis, as well as a limited cache of cancer-associated microRNAs (miRNAs). This proposal will provide functional annotation vis-a-vis the Hippo pathway in pancreatic cancer initiation and maintenance, using a variety of preclinical models. Specifically, in Aim 1, a role for Hippo pathway in pancreatic progenitor cell expansion will be studied using transgenic zebrafish with ectopic hYAP expression in the exocrine pancreas. The zebrafish studies will also elucidate whether common oncogenic and represser mutations found in human pancreatic cancer demonstrate genetic cooperation with hYAP in inducing pancreatic neoplasia. In Aim 2, modulation of Hippo signaling will be studied in mammalian systems. Specifically, Yap will be conditionally ablated in a compound transgenic mouse model of pancreatic neoplasia and the effects on natural history of the disease and immunophenotype of resulting pancreatic lesions studied. Further, a panel of isogenic human pancreatic cancer lines will be generated by engineering missense mutations of hFAT1 and hFAT4 at their endogenous loci, in order to study the biochemical mechanisms associating the membrane bound FAT protocadherins to intracellular events in the Hippo pathway. Lastly, in Aim 3, hYAP-regulated miRNAs will be analyzed for their ability to recapitulate or rescue the phenotype of aberrant Hippo signaling in pancreatic cancer.